It has been known that liver microsomal cytochrome P-450 proteins catalyze hydroxylations of steroid hormones, yet a specific cytochrome P-450 for the hydroxylation has not been characterized. Steroid hydroxylation activity (P-450) exhibits age- and sex- dependent expression. The expression of a male-specific testosterone 16Alpha-hydroxylase and a female-specific testosterone 15Alpha-hydroxylase in adulthood are predetermined by neonatal androgen ("neonatal imprinting"). A change or failure of expression of these hydroxylases would alter the steroid metabolism and, in addition, drug oxidations by these hydroxylases. The change or failure, therefore, may result in an increase in drug toxicity or change in fertility, reproductive function, or sex gland differentiation. To understand these biological phenomena surrounding neonatal imprinting, this Unit has been first in the world to purify the 15Alpha-hydroxylase to electrophoretic homogeneity, and has been continuing to purify testosterone 16Alpha-hydroxylase. The purified protein is 48,000 daltons, and the Soret peak of the reduced hemoprotein CO complex exhibits a maximum at 451 nm. This form of P-450 accounts for no more than 1% of total mouse liver P-450 proteins. Virtually 100% of mouse liver microsomal testosterone 15Alpha-hydroxylation activity can be accounted for by the purified 15Alpha-hydroxylase. By obtaining specific antibodies to the 15Alpha- and 16Alpha-hydroxylase P-450 proteins, and eventually their cDNA and genomic clones, we hope to understand the mechanism of neonatal imprinting of these P-450 proteins--How they are irreversibly "fixed" during the neonatal period and how these genes are regulated by hormones later in life.